Pancreatic cancer is the 4th leading cause of cancer fatalities in the United States both in men and women, with an overall 5-year survival rate of only 3-5%. As the current therapies offer very limited survival benefits, novel therapeutic strategies are urgently needed to treat this aggressive disease. Given the close relationship between the endocrine and exocrine pancreas, a unique feature of the pancreatic microenvironment is the potential influence of locally produced insulin on pancreatic cancer cell development. Crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling systems plays a critical role in the regulation of many physiological functions and in the pathogenesis of a variety of abnormal processes, including cancer cell proliferation. Despite the biological and clinical importance of this receptor crosstalk, the molecular mechanism(s) involved remain poorly characterized. This R21 application (in response to RFA, entitled "Pilot Studies in Pancreatic Cancer") is based on our recent findings showing that exposure of human pancreatic cancer cells to physiological concentrations of insulin rapidly enhances early cellular events, such as increase in [Ca2+]i elicited by Gq-coupled receptor agonists, including angiotensin II, bradykinin, bombesin and neurotensin. Using real-time imaging of changes in PtdIns(4,5)P2 hydrolysis and generation of Ins(1,4,5)P3 in single cells, we found that insulin enhances the rate and magnitude of these second messenger-generating responses. The insulin-induced potentiation of GPCR signaling is mediated through a rapamycin-sensitive, PI3- kinase/mTOR-dependent pathway. Our preliminary results also show that insulin and GPCR agonists synergistically stimulate DNA synthesis and anchorage-independent cell proliferation. We hypothesize that the potentiation of GPCR signaling by insulin through a mTOR-dependent pathway provides a mechanism by which insulin enhances the responsiveness of pancreatic cells to GPCR agonists, Metformin, a widely used drug for treatment of type 2 diabetes mellitus, negatively regulates mTOR function via AMPK activation. We hypothesize that metformin preferentially abolishes the crosstalk between insulin receptor and GPCR signaling. Indeed, preliminary results show that metformin inhibits the crosstalk between insulin receptor and GPCR signaling systems in Ca2+ signaling and DNA synthesis in human pancreatic cancer cells. Consequently, we propose to pursue two Specific Aims in this Grant application: 1) Characterize the mechanism(s) and growth-promoting effects of the crosstalk between insulin receptor and GPCR signaling in a panel of human pancreatic cancer cell lines;2) Characterize the inhibitory effects of metformin on the crosstalk between insulin receptor and GPCR signaling in vitro and in vivo. Given that metformin and rapamycin derivatives are FDA-approved drugs, these studies may pave the way for exciting and rapid translational research. PUBLIC HEALTH RELEVANCE: Conditions that enhance insulin production in the pancreas, including obesity and early stages of Type II diabetes, increase the risk of pancreatic cancer. An exciting new result shown in this application is that metformin, a drug widely used in the treatment of type II diabetes, inhibits a novel crosstalk between insulin receptor and Gq-coupled receptor signaling, blocks the growth of human pancreatic cancer cells and thus, can provide a novel therapeutic strategy for pancreatic cancer.